School of Chemistry - Theses
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ItemImaging the interactions of antimicrobial peptides with model and living cellular membrane systems( 2016)Since the discovery of penicillin over 80 years, the use of antimicrobial compounds has undoubtedly been one of the greatest contributions to modern science and medicine. Due to their use, misuse, and abuse over this time, numerous bacterial species have developed countermeasures to resist the effects of these antimicrobials. This rise in antimicrobial resistance has continued unabated since their first use, and now has the potential for devastating costs to humanity on a global scale; notably, mortality rates well into the millions over the coming decades. In turn, this has sparked interest in new therapeutic options not based on currently existing compounds. Antimicrobial Peptides (AMPs) are one such class of compounds. They can be found in virtually all forms of life, and act as the first-line- of-defence against a broad spectrum of pathogens at very low concentrations with limited resistance capacity. This makes AMPs extremely lucrative for future therapeutic design. Despite extensive investigations into their interactions, detailed mechanistic information about their behaviour with membranes remains elusive. Numerous models have been proposed to account for the observed behaviour of cell death, including pore-formation, detergent action, or some combination of the two. Mechanistic information regarding the progression of these models remains limited, however. In this work, we investigate the mechanistic behaviour of a model AMP with artificial and living bacterial membranes, primarily through the means of time- resolved fluorescence microscopy techniques. The antimicrobial peptide under study is an analogue of melittin; a well-characterised lytic peptide that displays significant activity to both eukaryotic and prokaryotic membranes. The analogue also bears a proline-to-lysine substitute around the centre of the peptide sequence, to which the fluorescent probe Alexa Fluor 430 is grafted. Using known photophysical information of the fluorescent peptide, information regarding the mechanistic progression of its interaction with membranes can be observed and deconvolved. This research progresses from developing a simple mechanistic understanding using model membrane systems, to increasingly complex living systems. In the liposome giant vesicle model, a slow two-step pore-formation mechanism was found to fit to the observed data, with major significance on the roles of aggregates. Leakage studies using the same liposome system found that the same pore-forming mechanism occurred in both unilamellar and multilamellar membranes, but with dramatically reduced pore stability in the latter. Similar experiments on E. coli bacteria revealed a more complex interaction that could not be adequately explained using either model systems. This finding has important ramifications for correlating information gained from model studies to living membranes. Finally, peptide interactions with Pseudomonas aeruginosa L-Forms were investigated in detail for the first time using super-resolution and fluorescence lifetime microscopies. The use of a biologically relevant environment was found to drastically alter the observed outcome of the peptide-membrane interaction, although the importance of peptide aggregates remains. Furthermore, little change in the fluorescence lifetime is observed over time, despite a clear time-resolved killing mechanism. The reasons for this, and the potential importance of the L-Form state, are explored in detail.